Autoproteolysis of a 45 kDa Human Shh precursor protein gives a 20 kDa N-terminal fragment that is responsible for normal hedgehog signalling and a 25 kDa C-terminal fragment involved in autoprocessing activity in which the N-terminal fragment is conjugated to cholesterol (Lee et al. Science 266 1528-1537 (1994) and Bumcrot et al. Mol. Cell. Biol. 15 2294-2303 (1995)).
Normally functioning Hedgehog (Hh) signaling specifies embryonic pattern by directing cellular differentiation and proliferation, which was first reported in Drosophila melanogaster (Nusslein-Vollhard et al. Roux. Arch. Dev. Biol. 193: 267-282 (1984)). Cellular responses to the secreted Hh polypeptide are mediated by two integral membrane proteins, Patched (Ptc) and Smoothened (Smo). Hh binds to the twelve transmembrane protein Ptc and hence reverses the Ptc-mediated suppression of the seven transmembrane protein Smo. This Smo activation then triggers a series of intracellular events, culminating in the stabilization of the transcription factor Cubitus interruptus (Ci) and the expression of Ci-dependent genes. These events are recapitulated during mammalian development and tumourigenesis through multiple protein homologues, including three distinct Hh family members [Sonic (Shh), Indian (Ihh), and Desert (Dhh)], two Ptc proteins (Ptch1 and Ptch2), and three Ci-like transcription factors (Gli1, Gli2, and Gli3). However, there is a single vertebrate homologue of Smo, which is implicated in all forms of Hh signaling by genetic analyses in Drosophila, mice, and zebrafish (Chen et al. PNAS 99(22): 14071-14076 (2002)).
Smo initiates a signal cascade causing the activation of Gli transcription factors and their subsequent nuclear translocation resulting in the control of transcription of target genes. Through a negative feedback loop, Gli influences transcription of Ptc and Hip 1 (hedgehog-interacting protein 1 (Hip1)) which inhibit the Hh pathway. The loss of control over the activation of the Hh pathway has been associated with an increasing range of cancers including those affecting the brain such as medulloblastoma (Romer and Curran, Cancer Res 65(12) 4975-4978 (2005)) and glioblastoma (Bar et al. Stem Cells 25(10):2524-33 (2007)); prostate cancer (Sanchez et al. PNAS 101(34) 12561-12566 (2004)); pancreatic cancer (Thayer et al. Nature 423 851-856 (2003)); non-small cell lung carcinoma (Yuan et al. Oncogene 26 1046-1055 (2007); small-cell lung cancer (Watkins et al. Nature 422 313-317 (2003)); breast cancer (Kubo et al. Cancer Res 64 6071-6074 (2004)); various digestive tract tumours (Berman et al. Nature 425 846-851 (2003)) and (Lees et al. Gastroenterology 129(5) 1696-1710 (2006)); basal cell carcinoma (Williams et al. PNAS 100(8) 4616-4621 (2003)); malignant melanoma (Pons and Quintanilla Clin Trans Oncol. 8(7) 466-474 (2006)); squamous cell carcinomas (Xuan et al. Mod Pathol. 19(8) 1139-47 (2006)); B-cell malignancies such as multiple myeloma and lymphomas (Dierks et al. Nat. Med. 13(8) 944-951 (2007); Peacock et al. PNAS 104(10) 4048-4053 (2007)); mesenchymal cancers such as chondrosarcoma (Tiet et al. Am. J. Pathol. 168(1) 321-330 (2006)), clear cell sarcoma of the kidney (Cutcliffe et al. Clin Cancer Res. 11(22):7986-94 (2005)) and rhabdomyosarcoma (Tostar et al. J. Pathol. 208(1) 17-25 (2006)); chronic myeloid leukaemia (Sengupta et al. Leukemia 21(5) 949-955 (2007)); endometrial carcinoma (Feng et al. Clin. Cancer Res. 13(5) 1389-1398 (2007); hepatocellular carcinomas (Huang et al. Carcinogenesis 27(7) 133401340 (2006)); ovarian tumours (Chen et al. Cancer Sci. 98(1) 68-76 (2007)).
It has also been found that Hh signaling regulates the expression of the ABC transporter proteins multi-drug resistance protein-1 (MDR1, ABCB1, P-glycoprotein) and (BCRP, ABCG2), and that targeted knockdown of MDR1 and BCRP expression by small interfering RNA partially reverses Hh-induced chemoresistance. This would suggest that the Hh pathway may be a target to overcome MDR and increase chemotherapeutic response (Sims-Mourtada et al Oncogene 26(38) 5674-5679 (2007)). The blockade of sonic hedgehog signal pathway was found to enhance the antiproliferative effect of EGFR inhibitors in pancreatic cancer cells (Hu et al. Acta Pharmacol Sin. 28(8) 1224-30 (2007)) and prostate cancer cells (Mimeault et al. Int. J. Cancer 118(4) 1022-31 (2006)).
The hedgehog pathway has also been associated to tumour regrowth after chemoradiotherapy and as a potential target to improve radiation response (Sims-Mourtada et al. Clin. Cancer Res. 12(21) 6565-6572 (2006)) and cyclopamine, a hedgehog pathway antagonist, increases the cytotoxic effects of paclitaxel and radiation in Hh expressing pancreatic cancer cells (Shafaee et al. Cancer Chemother. Pharmacol. 58(6) 765-70 (2006)).
It has also been reported that the inhibition of the Hedgehog signalling pathway may be of use for the treatment of a range of diseases related to inflammation, epithelial cell hyperplasia, fibrosis of tissue or immune disorders (Lamb et al. EP1183040). Inhibition of sonic hedgehog signaling has been reported to reduce chronic rejection and prolong allograft survival in a rat orthotopic small bowel transplantation model. Although acute graft rejection can be controlled by immunosuppressive agents, chronic rejection, which is characterized by arteriosclerosis in the donor organ vessels, is a major hurdle to long-term allograft survival. Graft survival in a rat orthotopic small bowel transplantation model was significantly prolonged after anti-Shh antibody treatment compared with the immunoglobulin G control (116 vs. 77.5 days). Collagen deposition and vascular occlusion in the mesentery were markedly reduced in recipients of the anti-Shh antibody (Chen et al. Transplantation 83(10) 1351-1357 (2007); Lamb et al. EP1183040B1).
It has also been reported that sFRP-1 is the downstream target gene of Hh signaling and that elevated expression of secreted frizzled related protein-1 (sFRP-1) following activation of the Hh pathway provides the molecular link for the inhibitory effect on Wnt signaling (He et al. J. Biol. Chem. 281(47)35598-35602 (2006)). Thus the modulation of Wnt signaling by antagonising Hh pathway through sFRP-1 could provide a method for the treatment of a range of diseases such as osteoporosis (Ai et al. Mol. Cell. Biol. 25(12) 4946-4955 (2005)) among others (Luo et al. Laboratory Investigation, 87, 97-103-(2007)).
Various inhibitors of the Hh pathway have been investigated, including the natural product cyclopamine, which is believed to act by binding to the heptahelical region of Smo. Additionally a number of synthetic small molecule antagonists of the Smo receptor have been reported in recent years: for a review see Kiselyov Anti-Cancer Agents in Medicinal Chemistry 6 445-449 (2006).